In producing commercially viable proteins, the ability of the microorganism to secrete the protein into the broth in bioactive form is important. However, there are many proteins encoded by genetically engineered DNA constructs which may not be secreted by the cells in which the DNA is expressed or which may not secrete the protein in bioactive form. If the protein is not secreted into the broth, downstream processing is necessary. This means that the cells must be harvested, the cell walls must be broken open, the desired proteins must be recovered in pure form and then such proteins must be chemically re-natured to restore their bioactivity. If the protein is secreted into the broth, but not in its bioactive form, the protein must be treated after secretion to restore its bioactivity.
Some cells and microorganisms carry out the biological equivalent of downstream processing by secreting proteins in bioactive form. The mechanism which directs the secretion of some proteins through the cellular exterior into the outside environment of the cell is not yet fully understood. For example, the species Streptomyces griseus secretes many extracellular proteins in bioactive form. It would be expedient if heterologous proteins of commercial value, whose bioactivity is a function of their particular three dimensional molecular structure, could be secreted from Streptomyces at the levels observed for natural extracellular proteins.
Some of the literature relating to genetically engineered DNA constructs has assumed that the production of a functional protein using the information contained in DNA was solved by decoding the DNA. This assumption was based on the principle that the information needed to specify the complex-three-dimensional structure of a protein molecule is contained in the primary amino acid sequence of the protein. However, Canadian Application No. 449,456 entitled Production of Active Proteins Containing Cystine Residues filed by Cangene Corporation on Nov. 1, 1985 illustrates that the bioactivity of certain proteins derived from genetically engineered DNA constructs is dependent upon the formation of correctly positioned disulphide bonds. A more effective means was sought than conventional methods for the expression of heterologous genes in a host cell or microorganism. Thus, that invention identified that heterologous proteins could be secreted from a host microorganism in bioactive form without resorting to downstream processing. The use of certain microorganisms in conjunction with an expression system facilitates the formation of disulphide bonds upon expression of the genetically engineered DNA construct. Bioactivity of engineered proteins having cystine residues as an integral and necessary portion of their active structure was achieved by using a regulatory nucleotide sequence selected from a cell or microorganism capable of expressing and excreting homologous disulphide-bonded proteins, the nucleotide sequence being operably linked to a second nucleotide sequence encoding a disulphide bond-containing heterologous protein. The regulatory nucleotide sequence encoded a protein which resulted in heterologous protein secretion from the cell or microorganism. The heterologous protein could be natural or designed.
In Canadian Patent Application no. 542,628 entitled Characterization and Structure of Genes for Protease A and Protease B from Streptomyces Griseus filed on Jul. 21, 1987 by Cangene Corporation, a homologous gene expression system was disclosed. That invention related to a regulatory nucleotide sequence which directed the secretion of Protease A and Protease B from Streptomyces griseus. Protease A and Protease B are naturally-occurring proteins in Streptomyces griseus, thus the terminology "homologous". That application disclosed the regulatory nucleotide sequence which was responsible for one type of homologous secretion in Streptomyces. A gene expression system responsible for homologous expression was useful in constructing various other expression systems for heterologous expression.
Granulocyte macrophage colony stimulating factor ("GM-CSF") is a protein which stimulates the production of white blood cells. GM-CSF holds great promise as a biopharmaceutical for use in association with cancer treatment to aid in the restoration of white blood cells. Naturally occurring GM-CSF is a glycoprotein containing 127 amino acids and two disulphide bonds. GM-CSF is present in only trace quantities in the natural human source, which has prevented detailed structural analysis of the naturally isolated protein. Thus, most of the structural data for the natural GM-CSF is obtained from analysis of the complementary DNA sequence and the expression of a complementary DNA clone in mammalian cells. The GM-CSF which is expressed in mammalian cells contains 127 amino acids and two disulphide bonds, and is present in different glycosylated forms ranging in size from 14 to 35 kilodaltons. Some forms of GM-CSF may contain two N-linked carbohydrate groups and/or three O-linked carbohydrate groups, which accounts for the apparent size heterogeneity.
Moonen, P. J., et al., 1987 (Proc. Natl. Acad. Sci. U.S.A.) a process is described for the production of GM-CSF by secretion from chinese hamster ovary cells. The GM-CSF is secreted as a 26-kilodalton glycoprotein which is biologically active. However, the biological activity is increased 20-fold by enzymatically removing the carbohydrate groups, indicating that an unglycosylated form of GM-CSF should be superior for clinical use.
In Ernst, J. F. et al., 1987 (Bio/Technol. 5:831-834) a process is described for the production of GM-CSF by secretion from the yeast Saccharomyces cerevisiae by using the alpha mating factor precursor. The GM-CSF is secreted as a heterogeneous mixture of glycoproteins ranging in size from 35 to 100 kilodaltons. Only a fraction of the secreted GM-CSF had been correctly processed from the alpha mating factor precursor. The specific biological activity of the glycosylated GM-CSF made in yeast and in mammalian cells was approximately the same. However, the structure of the attached carbohydrate groups of the GM-CSF produced in yeast were different from the natural carbohydrate groups of the GM-CSF produced in mammalian cells.
In Burgess, A. W., et al 1987 (Blood 58:43-51) a process is described for the production of an unglycosylated GM-CSF-like polypeptide from the cytoplasm of E. coli. The GM-CSF-like polypeptide as isolated from the E. coli cells, had an amino terminal methionine, and was reduced, denatured, and biologically inactive. The conversion of the biologically inactive GM-CSF-like polypeptide isolated from E. coli to a bioactive form required oxidative renaturation in vitro. The renatured GM-CSF-like polypeptide was still not equivalent to an unglycosylated form of GM-CSF due to the presence of an amino-terminal methionine in the E. coli produced protein.
The GM-CSF which is secreted by mammalian cells or yeast is bioactive, but glycosylated. The GM-CSF which is isolated from E. coli is unglycosylated, but not bioactive. Thus, the conventional processes for producing GM-CSF require expensive, time consuming, or inefficient downstream processing to convert the form of GM-CSF from the culture to the bioactive, unglycosylated GM-CSF which is preferred for clinical use.
Consequently, a need exists for an expression system which will provide bioactive protein, in particular bioactive GM-CSF, upon secretion. Such a protein product would be different as a structure of matter than conventional protein products since structure determines bioactivity.